In FY19, we have continued to address the role of TGF-beta in regulating the cancer stem cell (CSC) compartment using a novel functional imaging approach that we developed to allow visualization of this minority cell population in real time and in situ. Our lentiviral-based CSC reporter uses a synthetic promoter in which expression of a fluorescent protein is driven by the stem cell master transcription factors Oct4 and Sox2. Using this approach, we have developed methods that allow extended cell fate mapping of individual CSCs in 2D and 3D culture systems and we have an ongoing collaboration with the lab of Dr. John Condeelis at the Albert Einstein College of Medicine to perform intravital imaging of the CSC population in the primary tumor and at the lung metastatic site. The intravital imaging approach has allowed us to demonstrate that the CSCs are slow-moving, invasive cells in the primary tumor that are preferentially associated with the microanatomical structure (TMEM) that drives intravasation of tumor cells into the bloodstream. We have shown induction of a stem phenotype in non-stem tumor cells on contact with macrophages, and have characterized changes in CSC representation across the entire metastatic process, demonstrating peak CSC representation on early arrival at the metastatic site. We are now addressing the effect of TGF-beta on CSC biology in breast cancer model systems that show either tumor suppressor or pro-progression responses to TGF-beta. Importantly, we have shown that treatment with neutralizing anti-TGF-beta antibodies in vivo will increase the cancer stem cell population in two xenograft breast cancer models in which TGF-beta functions as a tumor suppressor, while decreasing it in a model in which TGF-beta has pro-progression effects. Furthermore, we have evidence that TGF-beta signal transduction is different in stem vs. non-stem cells. These results have important implications for the ongoing clinical development of TGF-beta pathway antagonists. Studies to understand the detailed mechanisms by which TGF-beta induces either an expansion or a reduction in the cancer stem cell population are ongoing. We are focusing on effects of TGF-beta on phenotypic plasticity and differential effects on self-renewing vs differentiating cell divisions. We do this through imaging of stem and non-stem cells early after arrival at the metastatic site in the lung, as well as in 3D culture in vitro. We are also employing integrated genomic and single cell approaches to address the molecular mechanisms underlying differential effects of TGF-beta on CSC and non-CSC compartments at early and late stages of the disease process. Understanding how CSCs are regulated in vivo will be critical to development of more effective cancer therapies, as these cells are largely resistant to existing therapeutic approaches.